Over the past ten years, 89Zr-labeled antibodies have emerged as extremely promising agents for the PET imaging of cancer. However, the current methodology used to create these radiopharmaceuticals prevents them from reaching their maximum clinical potential. Specifically, a crucial step in the synthesis of these constructs is the coupling of an amine-reactive, bifunctional variant of the 89Zr4+ chelator desferrioxamine (DFO) to the lysine conjugates of the antibody. Yet because antibodies have lysine residues distributed throughout their structure, it is impossible to control the molecular location of the conjugation reaction or the number of chelators attached to the antibody. This lack of site-specificity results in poorly defined, heterogeneous constructs with suboptimal immunoreactivity and in vivo behavior. To address these issues, we have developed a chemoenzymatic strategy for the site-specific radiolabeling of antibodies. This approach reproducibly creates homogenous, well-defined, stable, and highly immunoreactive 89Zr-labeled antibodies that have been shown to exhibit in vivo performance comparable - and often superior - to the randomly-labeled constructs currently employed in the clinic. This collaborative proposal describes the synthesis, preclinical validation, and first-in-human clinical translation of 89Zr-SSDT, a site-specifically labeled 89Zr-DFO-trastuzumab radioimmunoconjugate for the PET imaging of HER2-positive metastatic breast cancer. The central hypothesis of this work is that a site-specifically labeled 89Zr-SSDT radioimmunoconjugate will exhibit improved performance as a PET imaging agent for metastatic breast cancer compared to the non-site- specifically labeled 89Zr-DFO-trastuzumab construct currently employed in the clinic. Specific Aim 1, executed during Years 1-2 of the award period, will be focused on the synthesis, in vitro characterization, and in vivo investigation of two, different 89Zr-SSDT constructs in murine models of breast cancer, with the aim of identifying the single most promising 89Zr-SSDT for clinical translation. Specific Aim 2, executed during Years 2-3 of the award period, will be centered on the study of the in vivo toxicology and pharmacology of 89Zr-SSDT as well as the preparation and submission of an FDA Investigational New Drug application for the clinical trial. The goal of Specific Aim 3, executed during Years 3-5 of the award period, will be the first-in-human clinical trial of 89Zr-SSDT for the PET imaging of HER2-positive metastatic breast cancer. This 20-patient trial will be focused on the evaluation of the clinical safety and efficacy of 89Zr-SSDT as well as the determination of its in vivo performance relative to the non-site-specifically radiolabeled 89Zr-DFO-trastuzumab currently employed in the clinic. Ultimately, we believe that this clinical trial could have a tremendous impact on clinical cancer care, in the near term enabling safer, more sensitive, and more effective PET imaging of breast cancer patients and in the long term helping to usher in an era in which homogenous, site-specifically labeled radioimmunoconjugates become the standard of care.